Phosphorylated aktser473

A total of 45 μg of protein was loaded and resolved by SDS-PAGE on 15% polyacrylamide gels, and transferred to nitrocellulose membranes. Immunoblotting was performed using the following primary antibodies: phosphorylated Akt^SER473, GSK3β (Abcam, Cambridge, MA, USA); pan Akt, SOCS3, phosphorylated GSK3β^SER9, phosphorylated AMPKα^THR172, and total AMPKα (Cell Signaling Technology, Danvers, MA, USA). After incubation with either goat-anti rabbit (HRP) or goat-anti mouse (HRP) secondary antibody (Thermo Scientific Pierce, Rockford, IL, USA), the immunoreactive complexes were detected with enhanced chemiluminescence (ChemiDoc™ XRS, Bio-Rad, Hercules, CA, USA) and quantified by densitometry using ImageJ software.

Western blotting was performed as described in our previous study [44 (link)]. The antibodies used in the present study included those against FTO (1:1000; Abcam), GAPDH (1:20000; Proteintech), Akt (1:2000; Abcam), phosphorylated-Akt^Ser473 (1:2000; Abcam), phosphorylated-GSK3β (1:1000; Cell Signaling Technology), Cyclin D1 (1:20000; Abcam), c-Myc (1:1000; Abcam), and PDGFC (1:2000; Abnova). The grayscale of indicated protein was quantified by image analysis software (ImageJ 1.51e, NIH Image).

A total of 45 μg of protein was loaded and resolved by SDS‐PAGE on 15% polyacrylamide gels and transferred to nitrocellulose membranes. Immunoblotting was performed using the following primary antibodies: phosphorylated Akt^SER473, leptin (Abcam, Cambridge, MA); pan Akt, SOCS3, phosphorylated AMPKα^THR172, and total AMPKα (Cell Signaling Technology, Danvers, MA). After incubation with goat‐anti rabbit (HRP) secondary antibody (Thermo Scientific Pierce, Rockford, IL), the immunoreactive complexes were detected with enhanced chemiluminescence (ChemiDoc^™ XRS, Bio‐Rad, Hercules, CA) and quantified by densitometry using ImageJ software. Each western was normalized to a loading control sample. This positive control varied depending on the target protein of the blot. The specific controls were as follows: pAkt/total Akt (C2C12 myotubes treated with 100 nmol·L⁻¹ dose of humulin R to mimic an acute dose of insulin for 15 min prior to cell lysis), pAMPKα/total AMPKα (C2C12 myotubes treated with 1 mmol·L⁻¹ AICAR for 24 h prior to cell lysis), and SOCS3/leptin (C2C12 myotubes treated with 100 nmol·L⁻¹ dose of humulin R for 3 days to mimic a chronic dose of insulin prior to cell lysis).